ABSTRACT: Integrin ?4 (ITG?4) and paxillin are essential adhesion molecules in endothelial cell (EC) focal adhesions (FAs), structures critical for mechano-sensing and the bidirectional signal transduction between the EC cyto- skeleton and the cell-matrix interface. While central for adhesion stability in response to mechanical stress and inflammatory lung injury, the mechanistic basis for dynamic FA coordination during EC barrier dysfunction and barrier restoration remains a fundamental but unresolved question. ITG?4 and paxillin exhibit unique structural and regulatory features pertinent to lung vascular barrier regulation. ITG?4 is unique from other laminin-binding integrins by the presence of the prominent long cytoplasmic domain that interacts with cytoskeletal proteins including actin and plectin, a protein link between the actin cytoskeleton and plasma membrane intercellular junctions. In addition, we have generated highly novel data demonstrating that EC express several ITG?4 alternatively-spliced variants that appear to be involved in mechano-sensing and EC barrier regulation. Paxillin is a multi-domain adapter FA protein that recruits structural and signaling molecules to FAs in concert with cyto- skeletal rearrangement. Project #3's working model is that coordinate control of FA structures requires the dynamic involvement of ITG?4 and paxillin as well as key PPG cytoskeletal effectors (nmMLCK, cortactin, EVL, c-Abl) to efficiently assemble functional adhesion structures during EC barrier responses (peripheral cytoskeletal remodeling, lamellipodial formation, gap closure). We speculate that these EC responses are highly influenced by post-translational modifications (PTMs) and coding polymorphisms (SNPs). SA #1 will evaluate the influence of ITGB4 coding SNPs (and known pathological ectodomain SNPs) and ITG?4 PTMs in the plectln- binding cytoplasmic domain on EC barrier responses. SA #1 will also examine the influence of ITG?4 SNPs on the generation of the unique ITG?4 alternatively-spliced variants we have identified in mechanically-stressed EC as well as their contributions to EC barrier responses. Project #3 scientists have shown that paxillin participates in both human lung EC barrier dysfunction as well in agonist-induced barrier enhancement. SA #2 will explore this dual role of paxillin in EC cytoskeletal rearrangement and barrier regulation and conduct in depth structure/function studies including live cell imaging of mutant fusion proteins. The influence of paxillin SNPs and c-Abl-mediated paxillin PTMs on differential paxillin interactions with critical cytoskeletal effectors and EC barrier responses will be investigated. Finally, SA #3 will test the functionality of ITGB4 and PXN coding SNPs and critical ITG?4 and paxillin PTMs in HGF- and S1P-mediated EC barrier enhancement in in vivo preclinical models of ARDS and VILI (Core C). Together with Projects #1 and #2, these studies will: i) determine the molecular basis for the coordinate regulation of critical ITG?4 and paxillin structure/function regions; ii) yield important insights into dynamic FA control by ITG?4, paxillin and the cytoskeleton; and thus, iii) create actionable EC barrier regulators for highly translational intervention.